In utero hematopoietic stem cell transplantation (IUHSCTx) is a way of reliably and safely introducing a very small number of corrective cells which will be permanently identified as "self rather than foreign without administering immunosuppressives that are potentially more harmful than the disease itself. However, except for a few cases, IUHSCTx has not been successful for the treatment of other congenital diseases in which no immunodeficiency exists. In the absence of immunosuppression, only microchimerism (<0.3%) has been achieved in human fetal recipients or non-human primate models of IUHSCTx. These levels are too low for the correction of most diseases and have not been demonstrated to reliably predict tolerance. The reasons for these failures are unclear but may relate to the poor competitive capacity for donor HSCs in the intact fetal hematopoietic environment or possibly the existence of an unrecognized immunologic mechanism in the fetal innate immune system. Evidence exists for both possibilities; however, the experiments described in this application will specifically explore the innate immune barrier to allotransplantation in early gestation fetus. From our studies in mice, we have observed that engraftment following IUHSCTx correlates with the existence of a mismatch in class Ib antigen expression in the donor or host strain and expression of the corresponding inhibitory NK receptor in the other strain. The level of engraftment appears to be in the direction of a class Ib mismatch and does not follow the degree of class la disparity. These characteristics are reminiscent of KIR-mismatched haploidentical bone marrow transplantation. In these experiments, we hypothesize that a donor/host mismatch in Qa-1/NKG2 signaling and Qa-2 expression regulates the outcome of in utero hematopoietic stem cell transplantation in immunocompetent recipients. The developmentally linked pattern of nonpolymorphic class Ib ligands and NKG2 inhibitory receptors on fetal NK cells is highly conserved between primates and rodents. These striking similarities provide great translational relevance for the study of fetal innate immunity in pre-thymic mice. To evaluate our hypothesis we will specifically: 1) Characterize the effect of a Qa-1/NKG2 mismatch in the response to the prenatal transplantation of allogeneic fetal liver; and 2) Determine the functional significance of a Qa-2 mismatch in the prenatal transplantation of allogeneic fetal liver. The results of these novel experiments will guide future application of IUHSCTx and potentially broaden its application to congenital diseases such as thalassemia, sickle cell disease, cystic fibrosis, diabetes mellitus, and various metabolic diseases. Additionally, for the first time, we will probe the mechanisms regulating the innate immune responses regulating fetal allotransplantation. [unreadable] [unreadable] [unreadable] [unreadable]